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1 Patterns and Mechanisms of Sex Ratio Distortion in the 2 Collaborative Cross Mouse Mapping Population 3 4 5 Brett A. Haines*, Francesca Barradale†, Beth L. Dumont*,‡ 6 7 8 * The Jackson Laboratory, 600 Main Street, Bar Harbor ME 04609

9 † Department of Biology, Williams College, 59 Lab Campus Drive, Williamstown, MA 01267 10 11 ‡ Tufts University, Graduate School of Biomedical Sciences, 136 Harrison Ave, Boston MA

12 02111

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13 Running Title: Sex Ratio Distortion in House Mice 14 15 Key words: sex ratio distortion, Collaborative Cross, intergenomic conflict, sex chromosomes, 16 ampliconic genes, Slx, Slxl1, Sly, Diversity Outbred, house mouse 17 18 Address for Correspondence: 19 20 Beth Dumont 21 The Jackson Laboratory 22 600 Main Street 23 Bar Harbor, ME 04609 24 25 P: 207-288-6647 26 E: [email protected]

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27 ABSTRACT 28 29 In species with single-locus, chromosome-based mechanisms of sex determination, the laws of 30 segregation predict an equal ratio of females to males at birth. Here, we show that departures 31 from this Mendelian expectation are commonplace in the 8-way recombinant inbred 32 Collaborative Cross (CC) mouse population. More than one-third of CC strains exhibit significant 33 sex ratio distortion (SRD) at wean, with twice as many male-biased than female-biased strains. 34 We show that these pervasive sex biases persist across multiple breeding environments, are 35 stable over time, are not fully mediated by maternal effects, and are not explained by sex-biased 36 neonatal mortality. SRD exhibits a heritable component, but QTL mapping analyses and 37 targeted investigations of sex determination genes fail to nominate any large effect loci. These 38 findings, combined with the reported absence of sex ratio biases in the CC founder strains, 39 suggest that SRD manifests from multilocus combinations of alleles only uncovered in 40 recombined CC genomes. We speculate that the genetic shuffling of eight diverse parental 41 genomes during the early CC breeding generations led to the decoupling of sex-linked drivers 42 from their co-evolved suppressors, unleashing complex, multiallelic systems of sex 43 chromosome drive. Consistent with this interpretation, we show that several CC strains exhibit 44 copy number imbalances at co-evolved X- and Y-linked ampliconic genes that have been 45 previously implicated in germline genetic conflict and SRD in house mice. Overall, our findings 46 reveal the pervasiveness of SRD in the CC population and nominate the CC as a powerful 47 resource for investigating sex chromosome genetic conflict in action. 48 49 50 ARTICLE SUMMARY 51 We compiled breeding records from The Collaborative Cross (CC) mouse mapping population 52 to quantify the frequency and explore potential mechanisms of sex ratio distortion. Strikingly, 53 more than one-third of CC strains yield significantly sex-biased litters. These sex biases are not 54 mediated by environmental effects and are moderately heritable. We conclude that the 55 widespread sex ratio distortion in the CC manifests from multilocus permutations of selfish sex- 56 linked elements and suppressors that are only recovered in the recombinant CC strains. 57

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58 INTRODUCTION 59 60 In species with single locus sex determination, Mendel’s rules of inheritance predict an equal 61 number of males and females at birth. However, departures from this idealized expectation are 62 common in nature. Many species can manipulate offspring sex ratios based on prevailing 63 environmental conditions (Hamilton 1967; Nager et al. 1999; West and Sheldon 2002), including 64 season (Drickamer 1990), maternal diet (Rosenfeld et al. 2003), and resource availability 65 (Douhard 2017). Additionally, increasing maternal stress loads lead to skewed sex ratios in 66 many mammals (Linklater 2007; Helle et al. 2008; Ideta et al. 2009; Ryan et al. 2012). 67 68 Beyond environmental influences, sex ratio biases can also arise from diverse genetic 69 mechanisms. At one extreme, a segregating X-linked recessive lethal allele will be 70 disproportionately associated with male lethality and distort the population sex ratio toward an 71 excess of females. Although selection should rapidly eliminate such a hypothetical variant from 72 the population, many mutations exhibit sex-specific phenotypic effects (Karp et al. 2017) and 73 could, therefore, contribute to sex biases in live birth ratios (e.g., McNairn et al. 2019). In 74 addition, prior work from diverse natural and laboratory model systems has demonstrated that 75 sex-linked selfish elements can drive sex ratio distortion (SRD) by promoting the transmission of 76 their resident chromosome at the expense of the other sex chromosome (Wood and Newton 77 1991; Seehausen et al. 1999; Cocquet et al. 2012; Unckless et al. 2015; Lindholm et al. 2016; 78 Helleu et al. 2016; Zanders and Unckless 2019; Courret et al. 2019). 79 80 Under most circumstances, the presence of an X-linked (or Y-linked) selfish element will impose 81 an intense selective pressure for the rapid emergence of a suppressor on the other sex 82 chromosome, thereby restoring a balanced population sex ratio. Consequently, at any given 83 point, a population is expected to be at or near sex ratio parity due to the action of counteracting 84 drive and suppressor elements. However, because different populations evolve independent 85 drive-suppressor systems, outcrossing can disrupt co-adapted systems of alleles to unmask the 86 presence of cryptic sex ratio distorters. Indeed, SRD is frequently observed in inter-population 87 (James and Jaenike 1990; Merçot et al. 1995), intersubspecific (Macholán et al. 2008; Phadnis 88 and Orr 2009; Good et al. 2010; Cocquet et al. 2012; Kruger et al. 2019), and interspecific 89 hybrids (Dermitzakis et al. 2000; Tao et al. 2001). SRD is also typically associated with reduced 90 fertility (Phadnis and Orr 2009; Cocquet et al. 2010, 2012; Zanders and Unckless 2019), 91 implying that selfish drive elements often impose a fitness cost. 92 93 The house mouse (Mus musculus) sex chromosomes are crucibles of historical genetic conflict 94 between feuding drive elements on the X and Y. More than 90% of the house mouse Y 95 chromosome is comprised of ampliconic genes with X-linked homologous partners that are 96 theorized to reflect historical bouts of driver-suppressor evolution (Mueller et al. 2008; Soh et al. 97 2014). One such family includes the SYCP3-like gene family members, Slx, Slxl1, and Sly, 98 which are present at upwards of 50-100 copies per genome (Scavetta and Tautz 2010; Morgan 99 and Pardo-Manuel de Villena 2017). The Y-linked gene Sly and its X-linked homologs Slx and 100 Slxl1 are selfish drive elements that promote the transmission of their parent chromosome, 101 although the molecular mechanisms by which this distortion is rendered are not fully 102 understood. Knockdown or deletion of Sly results in over-transmission of the X-chromosome 103 and female biased-litters (Conway et al. 1994; Cocquet et al. 2009). Conversely, knockdown or 104 deletion of Slx/Slxl1 yields male-biased litters (Cocquet et al. 2012; Kruger et al. 2019). 105 Although there are striking differences in Slx/Slxl1 and Sly copy number among house mouse 106 subspecies, the ratio of X- to Y-linked copies within subspecies has been maintained in 107 stochiometric balance over evolutionary time, presumably as a result of selection for balanced 108 sex chromosome transmission (Scavetta and Tautz 2010; Morgan and Pardo-Manuel de Villena

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109 2017). As expected, experimental crosses between subspecies and wild-caught intersubspecific 110 F1 house mouse hybrids frequently sire sex-biased litters (Macholán et al. 2008; Good et al. 111 2010; Turner et al. 2012). 112 113 Regardless of whether it emerges from environmental or genetic causes, SRD imposes 114 profound impacts on populations. Departure from a 1:1 sex ratio can influence the rate of 115 population growth, modulate the degree of mate competition, and even modify life-history 116 trajectories (Hamilton 1967; Le Galliard et al. 2005; Székely et al. 2014). In addition, SRD can 117 cause relative levels of diversity and divergence across the sex chromosomes and autosomes 118 to deviate from their theoretical expectations (Ellegren 2009; Wilson Sayres 2018). Further, 119 given that many diseases differ in incidence between the sexes (Ober et al. 2008; Regitz- 120 Zagrosek 2012), even subtle shifts in the sex ratio can lead to profound changes in the disease 121 burden of a population. 122 123 Despite its critical importance for the conservation of biodiversity, population dynamics, and its 124 role in shaping the genomic architecture of heterogametic vertebrate sex chromosomes, the 125 underlying biological mechanisms of SRD often remain elusive. In cases of environmentally- 126 induced SRD, we typically lack a comprehensive mechanistic understanding of how information 127 gathered from the maternal environment is biochemically relayed to the reproductive track to 128 manifest SRD (Krackow 1995; Navara 2013). Moreover, the genetic mechanisms that fuel 129 intergenomic conflicts between the sex chromosomes and lead to SRD are currently understood 130 at limited molecular resolution (Bravo Núñez et al. 2018; Courret et al. 2019; Kruger and Mueller 131 2021). On-going efforts to address these key knowledge gaps would be well-served by the 132 availability of robust and reproducible animal models equipped with powerful genomic resources 133 and tools for genetic engineering. 134 135 Toward this goal, we performed an exploratory analysis of the Collaborative Cross (CC) mouse 136 population to describe the prevalence and define potential causes of SRD in a premiere 137 mammalian model system (Churchill et al. 2004). The CC is an 8-way recombinant inbred panel 138 of mice developed from eight genetically diverse parental strains: A/J, C57BL/6J, 129S1/SvImJ, 139 NOD/LtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. Five of these founder strains (A/J, 140 C57BL/6J, 129S1/SvImJ, NOD/LtJ, NZO/H1LtJ) are classical inbred mouse strains of 141 predominantly M. m. domesticus ancestry (Yang et al. 2007). CAST/EiJ, PWK/PhJ, and 142 WSB/EiJ are wild-derived inbred strains representing each of the three cardinal house mouse 143 subspecies (M. m. castaneus, M. m. musculus, and M. m. domesticus, respectively). The 144 contribution of genetic material from three divergent subspecies, each with variable Slx/Slxl1 145 and Sly copy numbers, led us to specifically hypothesize considerable scope for genetic SRD in 146 this multiparent mapping population. 147 148 We collated detailed breeding records from 58 genetically distinct CC strains maintained and 149 distributed by The Jackson Laboratory to define patterns of SRD across this strain resource. We 150 find that weak SRD is widespread in the CC. We integrate in-depth analyses of breeding 151 records with genomic and phenotypic analyses to test the potential action of multiple 152 mechanisms of SRD. Taken together, our findings underscore the untapped potential of the CC 153 mouse mapping population to serve as a tool for dissecting the complex genetic mechanisms 154 and environmental drivers of SRD.

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155 METHODS 156 157 Compilation of Collaborative Cross breeding records 158 Breeding records were obtained for 58 Collaborative Cross strains maintained in The Jackson 159 Laboratory’s Repository between January 2016 and July 2019. Eleven strains sired <100 pups 160 surviving to wean age during this time frame and were excluded from further analyses. The data 161 recorded for each live-born litter include: strain name, unique dam and sire identifiers, dam date 162 of birth, sire date of birth, date mating established, litter birth date, litter size at birth, litter size at 163 wean, and the number of weaned males and females. All CC breeding data are provided in 164 Table S1. Strain-level summaries of these breeding data are provided in Table S2. 165 166 Breeding records for CC mice maintained at the University of North Carolina Chapel Hill (UNC) 167 were obtained from the UNC Systems Genetics website 168 (http://csbio.unc.edu/CCstatus/index.py?run=availableLines). These data are also made 169 available in Table S3. 170 171 Breeding records for 43 F1 crosses between distinct CC strains (i.e., CC-RIX crosses) were 172 kindly shared by colleagues at The Jackson Laboratory and are provided in Table S4. 173 174 For brevity, we exclude the laboratory code from CC strain names in figures and tables 175 throughout this manuscript. Note that in all cases of such ambiguity, we are referencing CC 176 lines maintained at the Jackson Laboratory. 177 178 Estimating sex ratios and survival statistics 179 For most analyses, CC strain sex ratios were calculated as the proportion of females at wean. 180 Sex ratios for CC lines maintained at UNC are presented in public data as the ratio of females to 181 males at wean. Comparisons of CC strain sex ratios between the JAX and UNC breeding 182 centers utilize JAX CC strain sex ratios calculated per this alternative definition. 183 184 Neonatal survival was approximated as the fraction of pups born to a given strain that survive to 185 wean. We acknowledge that this estimate is potentially imprecise, as it is often difficult to 186 accurately count pups at birth and pups that were cannibalized shortly after birth are likely 187 missed in these tallies. Litter size at birth was used as a proxy for the in utero survival rate. 188 Although litter size is shaped by a multitude of factors, strains with smaller litters may 189 experience higher rates of embryonic lethality than strains with larger litter sizes, all else being 190 equal. 191 192 To estimate survival during early in utero development and throughout the neonatal period, we 193 devised an ad hoc metric that combined litter size at birth and survival to wean. Specifically, we 194 computed the median litter size at birth and median birth-to-wean survival rate across all CC 195 strains. For a particular focal CC strain, we then computed the difference between the strain- 196 specific litter size and the overall CC population-wide median litter size. Similarly, we calculated 197 the difference between the strain-specific survival rate and the overall median survival rate in 198 the CC population. We then summed these two values into a single measure of aggregate 199 strain-specific survival from conception to wean. 200 201 Linear modeling and statistical analyses of temporal changes in SRD 202 The sex of each weaned pup was coded as a binomial indicator and modeled as a function of 203 strain, birth month, and birth year using the glm function in RStudio (v. 1.3.1056). Post-hoc Wald 204 tests were used to determine whether any independent variables provide a significant

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205 explanatory effect. R code to recapitulate these findings is available as a supplementary 206 document (cc_srd_analysis.R) on FigShare. 207 208 Analyses of maternal condition and male reproductive phenotypes 209 Maternal body mass and body fat percentage estimates for CC strains were obtained from the 210 McMullan1 and McMullan3 datasets in the Mouse Phenome Database (Table S5; (Bogue et al. 211 2020)). We also accessed male reproductive phenotype datasets for CC (Lazear, Shorter3, 212 Shorter4) and parental inbred strains (Odet1; (Odet et al. 2015)) via the Mouse Phenome 213 Database (Table S6). Spearman Rank correlations and Mann-Whitney U-tests were used to 214 assess relationships between phenotypes and SRD. R code to replicate analyses of maternal 215 condition (maternalCondition.R) and reproductive phenotypes (ReproPhenotypeAnalysis.R) is 216 available on FigShare. 217 218 Broad-sense heritability estimation 219 We treated the sex ratio estimated from weaned pups born to individual CC mating units as 220 independent, within-strain replicate phenotype measures. Mating units producing fewer than 30 221 pups over their breeding history were excluded due to the high uncertainty in calculated sex 222 ratios. We then fit a one-way ANOVA model (sex ratio ~ strain) to estimate the broad-sense 223 heritability (H2) of the sex ratio at wean using the interclass correlation method (Rutledge et al. 224 2014): MSB − MSW 225 H2 = MSB + (푛 − 1)MSW 226 227 where MSB and MSW are the mean squares between and within strains, respectively. Despite 228 being expressed as a proportion, the distribution of sex ratio values across the CC population is 229 bell-shaped, and we confirmed that a square root transform of the data has no meaningful 230 impact on the magnitude of our H2 estimate. 231 232 QTL Mapping 233 Single QTL mapping was performed using the linear mixed model framework implemented in 234 the R/qtl2 package (Broman et al. 2019). Genotype data at 110,054 unique genomic positions in 235 each individual CC strain and the CC linkage map were accessed from Dr. Karl Broman’s 236 GitHub page at https://raw.githubusercontent.com/rqtl/qtl2data/master/CC/cc.zip. Raw 237 genotypes were converted to genotype probabilities by invoking the Carter-Falconer mapping 238 function and assuming an error probability of 0.002. Relatedness among CC lines was specified 239 via a kinship matrix using the leave-one-chromosome-out method. QTL significance thresholds 240 were computed from 1000 permutations of the data. 241 242 We tested for significant effects of the Y and mitochondrial chromosomes on SRD using one- 243 way ANOVA, treating the strain origin of the Y chromosome (or mitochondrial genome) as a 244 factor. 245 246 R code to replicate QTL mapping and testing for Y and mitochondrial effects is available on 247 FigShare (qtlMapping_sexRatio.R). 248 249 Read mapping and Structural Variant Discovery 250 Whole genome sequences for the completed CC strains were previously released for 251 community use (Srivastava et al. 2017; Shorter et al. 2019b). Fastq files for each CC sample 252 were downloaded from the ENA archive under Project PRJEB14673. Fastq files for five CC 253 lines (CC019, CC026, CC049, CC070, and CC076) were corrupted and we were unable to 254 pursue genomic analyses with these strains.

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255 256 Fastq files were first processed with fastp (version 0.20.1) to remove adaptor sequences and 257 evaluate QC metrics (Chen et al. 2018). Reads were then mapped to the mm10 reference 258 assembly using default parameter settings in bwa mem (version 0.7.17) (Li 2013). Optical 259 duplicates were marked using the MarkDuplicatesSpark command within GATK (version 260 4.1.8.1; (Van der Auwera and O’Connor 2020)). Structural variant (SV) discovery was 261 performed individually on each CC genome using manta (version 1.6.0; (Chen et al. 2016)). The 262 resulting SV vcf files were reformatted using the python script convertInversion.py supplied with 263 the manta distribution and subset to include only the non-ampliconic portion of the Y 264 chromosome (chrY:1-6.664Mb). The resulting vcf files were then uploaded into the Integrative 265 Genomics Viewer (IGV; version 2.8.0; (Robinson et al. 2011)) for manual inspection. 266 267 Estimation of gene copy number for ampliconic sex-linked families 268 Depth of coverage was computed in 1kb non-overlapping sliding windows using mosdepth 269 (version 1.18), ignoring read duplicates (Pedersen and Quinlan 2018). Coverage values were 270 then corrected for GC biases using a custom R script (compute_GC_depth_correction.R; 271 available on FigShare). Briefly, for each strain genome, we computed the average read depth 272 across autosomal regions with identical GC content, excluding outlier windows with >2 and 273 <0.333 the average autosomal coverage. We then used LOESS regression to fit a second- 274 degree polynomial (span parameter = 0.7) to the data to model the empirical relationship 275 between GC-content and average depth across the genome. The difference between the 276 genome-wide average read depth and the predicted read depth for a given GC-content value 277 corresponds to the average over- or under-representation of sequenced reads derived from 278 regions in that GC-content bin. These values were used as correction factors to adjust the 279 observed read depth in a given 1kb window based on its GC-content. GC-corrected read depths 280 were then standardized by the average genome-wide coverage to convert to copy number 281 estimates. Finally, data were compiled in bedGraph format for custom visualization in IGV. 282 bedgraph format files are provided on FigShare. 283 284 To estimate the copy number of the X and Y-linked ampliconic genes Slx/Slxl1, Sly, Sstx, and 285 Ssty1/2, we first identified the reference coordinates of all annotated paralogs from these genes 286 using the Ensembl Paralogues feature. To discover any additional unannotated paralogs, we 287 blated each annotated paralog sequence against the mm10 reference, retaining full-length hits 288 with >90% sequence identity to the parent sequence. Genomic coordinates for all ampliconic 289 genes are provided in Table S7. For each CC strain, we then summed the average read depth 290 across all paralogs to obtain an overall estimate of gene family copy number. 291 292 Copy number quantification by droplet digital PCR 293 Slx, Slxl1, and Sly copy number states were independently confirmed in a representative 294 sample of six CC strains (CC032/GeniUncJ, CC011/UncJ, CC003/UncJ, CC004/TauUncJ, 295 CC028/GeniUncJ, CC061/GeniUncJ) using droplet digital PCR (ddPCR). Genomic DNA was 296 isolated from spleen tissue using a Qiagen DNAeasy kit following manufacturer 297 recommendations. DNA was subsequently restriction digested with HaeIII for 60 minutes at 298 37C. DNA was then diluted 1:100 and 1:10 and combined with QX200 ddPCR EvaGreen 299 Supermix (BioRad) and custom-designed primers targeting either Slx/Slxl1, Sly, or the diploid 300 control Rpp30 locus (Table S8) per vendor protocols. The PCR reaction mixture was then 301 partitioned into droplets using a QX200 AutoDG Droplet Generator (BioRad). Emulsified 302 reactions were cycled on a C1000 Touch Thermocycler (BioRad) according to the following 303 program: 5 min initial denaturation at 95C; 40 cycles of 30s elongation at 95C, 30s of annealing 304 at 55C, and 60s elongation at 72C; and a final 15 min incubation at 4C to stabilize fluorescent 305 signals. Completed reactions were then held at 20C until removal from the thermocycler. Finally,

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306 reaction products were loaded into a QX200 Droplet reader and analyzed using QuantaSoft 307 Analysis Pro Software (v. 1.0.596; BioRad). Copy number estimates were standardized to 308 Rpp30 and averaged across a minimum of two technical replicates at each DNA concentration. 309 Final copy number estimates were then averaged across the two analyzed concentrations. 310 311 Animal Husbandry and Use Statement 312 Collaborative Cross strains were obtained from the Jackson Laboratory’s Repository and 313 housed in a low barrier room in accordance with an animal care protocol approved by The 314 Jackson Laboratory’s Animal Care and Use Committee (Protocol # 17021). Mice were provided 315 with food and water ad libitum. Sexually mature males were euthanized by exposure to CO2 at 316 10-14 weeks of age. 317 318 Testis Histology 319 Whole testes from three CC032/GeniUncJ males were fixed in Bouin’s fixative overnight at 4C 320 and then rinsed in a sequential ethanol series (25%, 50%, 3x 70% for 5 minutes each). Fixed 321 tissues were then submitted to the Histopathology Sciences Service at The Jackson Laboratory 322 for paraffin embedding, 5 m cross-sectioning, and regressive staining with Mayer’s 323 Hematoxylin and Eosin-Y. Slides were then scanned on a Hamamatsu NDP Nanozoomer at 40x 324 magnification and analyzed using NDP.view2 software. 325 326 Two independent cross-sections from two of the three biological replicates were then scored for 327 the following testis histology phenotypes: the total number of tubules per cross-section, total 328 cross section area, the number of tubules with vacuoles, the number of tubules with eosinophilic 329 cells (a proxy for cell death), and the cross-sectional area of 50 representative tubules. Testis 330 cross-sections from the third CC032/GeniUncJ replicate exhibited an unusually high fraction 331 (>50%) of vacuolized tubules harboring no post-meiotic cells (Figure S1). Inclusion of data from 332 this sample vastly skewed the estimates from other replicates and was dropped from the 333 analysis. Due to the limited number of surveyed replicates, we cannot exclude the possibility 334 that this vacuolized seminiferous tubule phenotype affects a reproducible subset of 335 CC032/GeniUncJ males, as previously reported for inbred WSB/EiJ males (Odet et al. 2015). 336

337 338 Figure S1. Hematoxylin and Eosin-Y stained testis cross section from a CC032/GeniUncJ male 339 at ~40x magnification. A high proportion of seminiferous tubules exhibit large vacuoles and 340 germ cell loss. 341

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342 343 Sperm whole chromosome painting 344 Sperm specimens were prepared for whole chromosome painting as described (Sarrate and 345 Anton 2009; Dumont 2017). Sperm were passively isolated from the caudal epididymis in a drop 346 of sterile PBS at room temperature, then concentrated by centrifugation and gradually 347 resuspended in ~1mL of Carnoy’s fixative. Several drops of fixed sperm were then spread 348 across a cleaned glass slide and allowed to air-dry. Slides were then rinsed in two successive 349 washes of 2x SSC for 3 min each, dehydrated in a sequential ethanol series (70, 90, 100% for 2 350 minutes at each concentration), and air-dried. Next, slides were washed in dithiothreitol solution 351 (5 mM 1,4-dithiothreitol, 1% Triton X-100, and 50 mM Tris) to decondense sperm DNA and 352 rinsed again in two consecutive washes of 2x SSC, dehydrated in a sequential ethanol series 353 (70, 90, 100%), and air-dried. 354 355 Sperm DNA was denatured in 70% formamide/2x SSC at 78C for 5 min. Slides were then 356 processed through a sequential ethanol series (70%, 85%, 100% for 1 min each dilution) and 357 air-dried. Simultaneously, Texas-Red labeled X chromosome and FITC-labeled Y chromosome 358 probes (Cytocell) were denatured for 10 minutes at 80C per vendor recommendations. Sperm 359 slides were then painted with a total volume of 10 L of denatured probes, a cover slip was 360 applied to the hybridized area, and sealed in place with rubber cement. Hybridization reactions 361 were allowed to process for ~48 hr at 37C. 362 363 After removing coverslips, slides were washed in 0.4x SSC/0.3% NP-40 at 74C for 2 minutes, 364 and 2xSSC/0.1% NP-40 at room temperature for 1 minute. After air-drying, slides were mounted 365 in ProLong Gold antifade media with DAPI (Invitrogen) and cover slipped. 366 367 Approximately ~1400 painted sperm cells were imaged on a Leica DM6 B upright epifluorescent 368 microscope equipped with GFP and Texas Red fluorescent filters and a cooled monochrome 369 2.8-megapixel digital camera. Images were post-processed and analyzed in the Fiji software 370 package (Schindelin et al. 2012). Individual sperm were scored as carrying an X or Y 371 chromosome based on fluorescent signal (Table S9). As no significant difference in the 372 frequency of X- versus Y-bearing sperm was noted in an initial sample, we did not perform 373 experiments on additional biological replicates or dye-swap probe combinations. 374 375 Sex ratio distortion in the Diversity Outbred population 376 Breeding records for the 175 Diversity Outbred (DO) breeding lineages were obtained from the 377 supplemental material of (Chesler et al. 2016) and are available in Table S10. These records 378 detail the parentage of litters born over 17 continuous generations of outbreeding (G6-G22). To 379 identify DO lineages siring sex-biased litters, we employed binomial tests to ask whether the sex 380 ratio of weaned pups born to females (or males) from each DO breeding lineage deviated from 381 the expected 1:1 ratio. An R script for reproducing this analysis is available on FigShare 382 (do_srd_analysis.R). 383 384 Data availability 385 The authors state that all data necessary for confirming the conclusions presented in this article 386 are represented fully within the article and supplemental material. R scripts for reproducing 387 analyses and figures are available on FigShare. VCF files with structural variant calls are 388 available upon reasonable request from the corresponding author. 389 RESULTS 390 391 Widespread Sex Ratio Distortion in the Mouse Collaborative Cross

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392 We collated breeding records from the Collaborative Cross mouse colony maintained in The 393 Jackson Laboratory’s Repository (Table S1). These records summarize the breeding 394 performance of 58 inbred CC strains organized into 3,890 independent mating units that 395 produced 54,034 pups between January 2016 and May 2019 (median = 874 pups per strain). 396 Eleven strains produced fewer than 100 pups during this time period and were excluded from 397 further analysis. Remarkably, 18 of the remaining 47 CC lines (38%) sired progeny with a 398 significant departure from the expected 1:1 sex ratio (uncorrected binomial P < 0.05; Figure 1). 399 Of these 18 strains, 12 are significantly male-biased, whereas six produce an excess of 400 females. This finding aligns with the significant, albeit slight, skew toward males in the 401 aggregate CC population breeding records (21,469 males versus 20,439 females; two-sided 402 binomial P = 4.99x10-7). The most significantly female-biased strain, CC065/UncJ, produces 403 67.4% females (n = 264; Binomial Test P = 1.54x10-8). CC032/GeniUncJ is the most male- 404 biased strain, siring 68.6% males at wean (n = 927; P = 2.83x10-30). 405 406 Statistical power to detect a significant departure from the expected Mendelian sex ratio is a 407 function of sample size. Many CC strains produced a modest number of pups over the survey 408 period, limiting our ability to detect weak SRD. Considering only those strains with >500 pups 409 (corresponding to ~60% power to detect a 45%:55% skew in the sex ratio; Figure S2), the 410 percentage of strains with significant SRD increases to 50%. We conclude that mild SRD is 411 pervasive in the CC reference mapping population, with a few strains showing extreme biases in 412 offspring sex ratios. 413

0.8

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male−biased n

a CC065 female−biased

e

W

t 0.6 CC080

a CC058 CC026

e

l CC084 CC013 a CC002

m CC006 CC012

CC068 CC040 e

F CC043 CC060

CC001

n CC009

o CC003

i 0.4 CC028

t

c a

r CC032 F

0.2 0 1000 2000 3000 Number of Weaned Pups 414 415 416 Figure 1. Sex ratio distortion in the realized Collaborative Cross population. Sex ratio is 417 expressed as the fraction of females at wean. The gray curved lines denote the level of noise 418 about the Mendelian expectation of 0.5 (dashed horizontal black line) that can be expected for a 419 given sample size due to binomial sampling error. Strains producing significantly male- and 420 female-biased litters are color-coded blue and red, respectively. 421

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1 0.9 0.8 0.7

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Percent Deviation From Sex Ratio Parity 422 423 424 Figure S2. Statistical power to detect sex ratio distortion using a two-way binomial test. Colors 425 and plotting shapes denote different sample sizes. 426 427 428 Testing the Stability of SRD to Environmental Influences 429 Seasonal fluctuations in external temperatures can influence offspring sex in captive laboratory 430 house mouse populations (Drickamer 1990). To test for seasonal and larger-term temporal 431 effects on SRD in the CC, we modeled the sex of each weaned pup as a binomial outcome of 432 strain identity, birth month, and birth year. Neither birth month nor birth year provide significant 433 predictive power in this model (birth month, Wald Test P = 0.74; birth year, Wald Test P = 0.17). 434 These findings are recapitulated on a per strain basis: there is no evidence for variation in sex 435 ratio from month-to-month within strains (Fisher’s Exact Test, P > 0.05; Table S11). 436 CC028/GeniUncJ and CC084/TauJ show slight variation in sex ratio from year-to-year (Figure 437 S3; Fisher’s Exact Test; PCC028=0.0365 and PCC084=0.0500), although these effects are modest 438 and do not remain significant after correcting for multiple testing. 439

440 441 Figure S3. The fraction of females at wean exhibits mild variation over time for 442 CC028/GeniUncJ and CC084/TauJ. Error bars correspond to 95% binomial confidence

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443 intervals. Sex ratios are estimated with high precision for some strain-year combinations, and 444 confidence intervals are masked by the plotting characters. Samples with significant male- and 445 female- sex biases are color coded blue and red, respectively. 446 447 448 To understand whether housing environment influences SRD in the CC, we next assessed the 449 concordance of JAX CC strain sex ratios with those of their sister-strain counterparts maintained 450 at an independent mouse facility at University of North Carolina (UNC) Chapel-Hill. Overall, 451 there is excellent concordance of the strain sex ratios between these two locations (Spearman’s 452 Rho = 0.760, P = 1.97x10-8; Figure 2). Notably, CC032 and CC065 are the most strongly male- 453 and female-biased strains, respectively, regardless of facility. Despite this overall agreement, 454 there are minor exceptions. For example, two strains that produce slightly male-biased strains at 455 JAX − CC060/UncJ and CC002/UncJ − fail to produce male-biased litters at UNC. These slight 456 discrepancies are likely attributable to binomial sampling error, rather than the accumulation of 457 independent mutations with effects on offspring sex ratio in the JAX and/or UNC colonies (i.e., 458 strain drift) or strain-by-environment interactions. 459

CC065

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0.5 1.0 1.5 2.0 2.5 JAX Sex Ratio 460 461 Figure 2. Sex ratios of CC strains maintained at JAX and UNC. Sex ratios on both axes are 462 expressed as the number of females to the number of males at wean. Strains that are 463 significantly female- and male-biased at JAX are color-coded red and blue, respectively. The 464 dashed gray diagonal line represents y=x. 465 466 467 Sex ratio is not influenced by maternal effects or breeding performance 468 Maternal condition has been shown to modulate live-birth sex ratios in many organisms (Nager 469 et al. 1999; Love et al. 2005). Using publicly available CC mouse phenotypes (Bogue et al. 470 2020; Table S5), we examined the relationship between sex ratio and two proxies of overall 471 maternal health: adult female body mass and body fat percentage. Sex-biased strains do not 472 have significantly different body mass or body fat percentages compared to sex-balanced 473 strains (Mann-Whitney U-Test; P > 0.05; Figure S4). Similarly, there is no significant difference 474 in body mass between female-biased strains and sex-balanced strains, or between male-biased 475 strains and sex-balanced strains (Mann-Whitney U-Test; P > 0.05). Although we find no

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476 significant link between these two metrics of overall maternal condition and offspring sex ratio, 477 we acknowledge that female-specific estimates of condition, rather than the strain-wide 478 estimates employed here, are most appropriate for rigorously testing this possible explanation 479 for SRD. 480

481 482 Figure S4. Relationship between sex ratio bias and mean adult female (A) body weight and (B) 483 body fat percentage. Each point corresponds to a single CC strain. CC strains are designated 484 as male- or female-biased based according to per-strain binomial tests (P < 0.05). 485 486 487 We next considered the possibility that random, non-genetic maternal effects influence offspring 488 sex in the CC. In the majority of CC strains, offspring sex does not vary from dam-to-dam within 489 a strain (Fisher’s Exact Test; P>0.05; Table S12). We do observe slight fluctuations in sex ratio 490 across breeding dams in CC004/TauUncJ (P = 0.013), CC061/GeniUncJ (P = 0.008), and 491 CC068/TauUncJ (P = 0.033), although these effects are not significant after accounting for 492 multiple testing (Figure S5; 42 tested strains, Bonferroni adjusted P = 0.0012).

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493 494 Figure S5. Dam identity exerts a weak influence on offspring sex ratios in CC004/TauUncJ, 495 CC061/GeniUncJ, and CC068/TauUncJ. For each strain, the sex ratio of animals sired by each 496 dam (or pair of dams, in the case of trio matings) is plotted as the fraction of females at wean. 497 Dams producing significantly female- or male-biased litters are denoted by the red and blue 498 points, respectively. Error bars correspond to 95% confidence intervals calculated from the 499 binomial distribution. 500 501 502 Prior work has uncovered significant effects of parental age and litter parity number on 503 mammalian sex ratios (Huck et al. 1988). Parental age at litter birth, litter size, and the number 504 of litters born to each mating unit vary among CC strains (Table S2), prompting us to explore 505 whether these variables contribute to the observed SRD. We modeled the sex of each weaned 506 pup as a binomial outcome of strain identity and either dam or sire age. Parental age does not 507 offer significant explanatory power in this model (P > 0.05). Similarly, litter size and litter number 508 do not impact estimated sex ratios (P > 0.05). 509 510 Sex ratio distortion is independent of maternal genotype 511 Different maternal genotypes provide distinct uterine environments for early development and 512 could modify sex ratios in crosses involving sires from a common strain. We leveraged breeding 513 data from crosses between distinct CC strains (i.e., CC-RIX crosses) carried out by colleagues

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514 at The Jackson Laboratory to address whether maternal genotype influences CC sex ratios. In 515 total, we surveyed data from 43 CC-RIX crosses profiling 24 different CC strains as sires (Table 516 S4). If sex ratio is strictly determined by the paternal transmission of X- versus Y-bearing sperm, 517 then the sex ratios of litters sired by males from different CC strains should be independent of 518 dam genotype. For each of the 24 CC sire strains included in this CC-RIX dataset, we asked 519 whether offspring sex varies as a function of dam strain identity. Although small sample sizes 520 limit our power (range: 13-103 progeny per CC-RIX cross; median = 31), we find no consistent 521 evidence for maternal genotype-dependence of sex ratios in the majority of the CC-RIX strains 522 tested (Fisher’s Exact Test P > 0.05; Table S13; Figures S6, S7). Three CC strains are 523 exceptions, with marginal maternal genotype dependence on offspring sex ratios: 524 CC027/GeniUncJ (P = 5.2x10-4), CC042/GeniUncJ (P = 0.038), and CC060/UncJ (P = 0.010) 525 (Figure S6). Notably, CC027/GeniUncJ males, when mated to CC027/GeniUncJ or 526 CC011/UncJ females, produced sex-balanced litters, but yield female-biased litters in crosses to 527 CC037/TauUncJ dams and male-biased litters in crosses to CC002/UncJ dams (Figure S6B). 528 Similarly, CC042/GeniUncJ males produce litters with a slight female bias in crosses with either 529 CC042/GeniUncJ or CC001/UncJ dams, and male-biased progeny in crosses to 530 CC005/TauUncJ females (Figure S6A). However, we caution that only the maternal effects in 531 CC027/GeniUncJ remain significant after correction for multiple tests. Overall, these findings are 532 in broad agreement with the absence of significant maternal genotype effects on sex ratios in 533 the eight parental founder strains of the CC (Shorter et al. 2019a). 534 535 In summary, we find no evidence that season, housing environment, dam identity, parental age, 536 litter number, litter size, maternal condition, or maternal genotype systematically influence sex 537 ratios across the CC strains. Based on these findings, we conclude that the sex ratio of a given 538 CC strain is likely an intrinsic, biological property of that strain, rather than a plastic response to 539 environmental factors or mediated via parental effects. 540

541 542 Figure S6. CC males from strains (A) CC042/GeniUncJ, (B) CC027/GeniUncJ, and (C) 543 CC060/UncJ sire litters with variable sex ratios depending on the genetic background of the 544 dam. Error bars correspond to 95% confidence intervals calculated from the binomial 545 distribution. Dam strains producing significantly male- or female-biased litters are color-coded 546 blue and red, respectively.

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Number of Pups Weaned 547 548 Figure S7. Sex ratio distortion in CC-RIX mice. The horizontal dashed line corresponds to the 549 expected 1:1 sex ratio. The gray solid curves delimit the range of sex ratio variation expected 550 due to binomial sampling for a given sample size. Crosses that yield significantly female- or 551 male-biased litters are shown in red and blue, respectively. Point labels specify the associated 552 CC-RIX cross in the format: dam x sire. 553 554 555 Evaluating sex differences in survival as a potential mechanism of sex ratio distortion 556 SRD can arise along a continuum of developmental timepoints ranging from differences in the 557 viability or fertilization efficiency of X- versus Y-bearing sperm to sex differences in post-birth 558 survival. If SRD stems from sex differences in survival during in utero development, more 559 extremely sex-biased strains should yield smaller litters. In contrast to this expectation, there is 560 no correlation between litter size and the absolute deviation from sex ratio parity in the CC 561 population (Figure 3A; Spearman’s Rho = -0.036, P = 0.819; analysis restricted to breeding 562 pairs only, to the exclusion of breeding trios and harem breeding units). Indeed, several sex- 563 biased strains - including CC013/GeniUncJ, CC060/UncJ, CC006/TauUncJ, and CC001/UncJ - 564 are among the most fecund CC lines. Similarly, sex differences in survival from birth to wean 565 are not correlated with SRD (Figure 3B; Spearman’s Rho = -0.0817, P = 0.601). 566 567 Mechanisms that contribute to increased rates of strain death in utero may also lead to 568 increased death rates in neonates. Consistent with this possibility, there is a significant positive 569 correlation between litter size and birth-to-wean survival rate; strains with larger litters at birth 570 have lower neonatal death rates (Figure 3C; Spearman’s Rho = 0.426, P = 0.005). We 571 combined these two measures of survival during pre- and post-natal development into a single 572 statistic that summarizes strain variation in survival from conception to wean (see Methods). 573 Again, we find no significant correlation between this composite survival statistic and the 574 magnitude of SRD (Figure 3D; Spearman’s Rho = -0.0467, P = 0.766). 575

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576 The absence of an overall association between survival in early development and SRD 577 suggests that mortality in early life does not provide a simple, unifying explanation for SRD in 578 the CC. However, it is noteworthy that two of the 18 significantly sex-biased strains are among 579 the 20% of CC strains with the lowest survival rates (CC026/GeniUncJ and CC040/TauUncJ; 580 Figure 3D). Sex differences in early development may contribute to SRD in certain strains, and 581 these findings motivate further work to dissect the developmental mechanisms of potential sex- 582 specific mortality in these lines. Nonetheless, survival differences are unlikely to explain SRD in 583 the majority of sex-biased strains.

584 585 Figure 3. Correlations between neonatal survival, litter size, and sex ratio distortion. Sex ratio 586 distortion is not significantly correlated with average litter size (A) or birth to wean survival rate 587 (B). Average litter size at birth and survival to wean are positively correlated (C). The strength of 588 sex ratio distortion is not correlated with aggregated in utero and birth-to-wean survival. Points 589 corresponding to strains with significant female- and male-bias are color-coded red and blue, 590 respectively. 591 592 593 No Evidence for Single Locus Mechanisms of Sex Ratio Distortion 594 Our extensive analyses of possible non-genetic explanations for sex ratio variation in the CC 595 turned up no compelling explanations, suggesting that sex ratio variation likely carries a genetic 596 basis. We utilized sex ratio estimates from independent breeding units within each CC strain as 597 biological replicates to compute the relative proportion of variation in the sex ratio that is due to 598 within versus between strain differences (i.e., broad sense heritability, H2; see Methods).

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599 Despite considerable binomial noise in sex ratio estimates per breeding unit, the sex ratio is 600 modestly heritable in the CC (H2 = 0.263). 601 602 To attempt to map genomic loci contributing to this heritable variation in SRD, we carried out a 603 genome-wide QTL scan in the CC. No autosomal or X-linked loci reached the genome-wide 604 threshold for significance (Figure 4A). Similarly, we find no effect of the Y chromosome or 605 mitochondrial haplotype on SRD (one-way ANOVA, P > 0.05; Figures 4B and 4C). Although 606 the small number of CC strains severely limits mapping power, we conclude that very large- 607 effect, single-locus modifiers of SRD are not likely segregating in the CC population. 608 609 This conclusion is bolstered by the observation that sex ratios in seven of the eight inbred CC 610 founder strains do not deviate from the expected 1:1 ratio of males:females (Shorter et al. 611 2019a). Founder strain 129S1/SvImJ produces a slight bias toward females (52.5%), but this 612 distortion is mild compared to that observed in the most extreme CC strains. In conjunction with 613 the overall absence of evidence for environmental or parental effects on SRD, these findings 614 raise the possibility that multilocus allelic combinations only uncovered in recombinant CC 615 genomes contribute to the widespread SRD in this multiparent mapping population. 616 Unfortunately, the modest number of realized CC strains effectively bars the application of 617 unbiased pairwise scans for such interacting loci. 618

619 620 Figure 4. Mapping single locus modifiers of SRD in the CC. (A) Genome wide scan for loci 621 influencing SRD in 49 CC strains. Horizontal lines correspond to the genome-wide permutation-

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622 derived thresholds for the autosomes and X chromosome. Fraction of females at wean for CC 623 strains partitioned by the parental strain origin of the Y chromosome (B) and mitochondrial (C) 624 haplotype. Strains with significantly male- and female-biased sex ratios are color-coded blue 625 and red, respectively. 626 627 628 Structural mutations at sex determination genes are unlikely to mediate SRD in the CC 629 Structural mutations encompassing key sex determination genes can lead to disparities 630 between phenotypic sex and sex chromosome genotype and could, potentially, manifest as 631 SRD. In most mammals, including house mice, sex is determined by the expression of a Y- 632 linked gene, Sry, in the undifferentiated gonad. Deletion or translocation of Sry from chrY 633 represent established genetic mechanisms for sex reversal in mammals (McElreavy et al. 1992; 634 Goodfellow and Lovell-Badge). 635 636 To explore the possibility that phenotypic sex is not a reliable indicator of sex chromosome 637 transmission in the CC, we scanned CC whole genome sequences (Srivastava et al. 2017) for 638 read mapping signatures consistent with structural mutations spanning Sry (see Methods). We 639 uncovered no evidence for translocations or deletions encompassing the Sry locus in any CC 640 strains. However, unexpectedly, we find that nearly all strains with the NOD/ShiLtJ Y 641 chromosome carry a ~200kb duplication spanning the complete Sry coding region (Figure S8). 642 CC003/UncJ is a single, notable exception: despite carrying a NOD/ShiLtJ Y chromosome, Sry 643 is present as a single copy gene. Invoking parsimony, we conclude that a deletion of the 644 duplicate Sry copy likely occurred during inbreeding of CC003/UncJ. A recent analysis of SNP 645 array data in diverse mice identified a larger, 2.9 Mb Sry-spanning duplication in C3H/HeJ mice 646 (Sigmon et al. 2020). The discovery of two independent Sry-spanning duplications in the 647 classical inbred strains and a putative de novo deletion in CC003/UncJ suggests that this locus 648 is inherently predisposed to recurrent genomic rearrangements and motivates further 649 investigation into structural genetic diversity at this critical developmental regulator in house 650 mice. 651 652 SRY activates the transcription of a second gene, Sox9, which in turn induces the testis 653 developmental program. Duplication (deletion) of Sox9 and/or its upstream regulatory elements 654 can lead to constitutively high (low) levels of Sox9 expression, providing a second mechanism 655 for sex reversal in mammals (Foster et al. 1994; Gonen et al. 2018). Interestingly, while we find 656 no evidence for duplication of Sox9 itself, we observe a ~1kb duplication and a ~2kb deletion 657 within the distal upstream Sox9 regulatory region that are specific to animals carrying the 658 NOD/ShiLtJ haplotype in these regions (Figure S9). These structural mutations do not span any 659 annotated regulatory elements in the mm10 reference genome, but it is tempting to speculate 660 that one or both may function to maintain native Sox9 expression levels in the face of potentially 661 increased SRY dosage driven by the Sry-duplication present in this strain. 662 663 It is unlikely that the NOD/ShiLtJ-specific SVs documented here are associated with an 664 appreciable rate of sex reversal. NOD/ShiLtJ has served as a prominent mouse model of 665 autoimmune disorders for more than 40 years, with no cases of sex reversal documented in this 666 strain or in crosses involving this strain (including the CC), to our knowledge. In addition, Sry 667 duplications are relatively common in rodent systems, and are not generally associated with sex 668 reversal (Nagamine 1994; Lundrigan and Tucker 1997; Bullejos et al. 1999). Finally, half of the 669 sex-biased CC strains do not carry the NOD/ShiLtJ haplotype at either Sox9 or Sry (Table S14), 670 necessarily assigning causality of SRD to other mechanisms. In summary, although we find 671 novel structural rearrangements spanning the Sry sex determination gene and within the 672 putative regulatory region of its upstream signaling target, Sox9 (Figures S8 and S9), these

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673 mutations seem unlikely, at face-value, to induce high rates of sex reversal and contribute to the 674 widespread SRD in the CC population. 675

676 677 678 Figure S8. Estimated genomic copy number in 1kb windows across the Sry locus on the short 679 arm of chrY for a sample of CC lines. CC lines carrying the NOD/ShiLtJ Y chromosome are 680 depicted in dark blue. Other CC strains are color coded by the parental origin of their Y 681 chromosome: yellow (A/J), gray (C57BL/6J), pink (129S1/SvImJ), light blue (NZO/HlLtJ), green 682 (CAST/EiJ), red (PWK/PhJ), and purple (WSB/EiJ) The faint horizontal dashed line on each plot 683 corresponds to the expected CN=1 state.

684

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685 Figure S9. Estimated genomic copy number in 1kb windows across the Sox9 locus on chr11 for 686 a sample of CC lines. CC lines carrying the NOD/ShiLtJ haplotype at this locus are depicted in 687 dark blue. Other CC strains are color coded by the parental origin of their chromosome: yellow 688 (A/J), gray (C57BL/6J), pink (129S1/SvImJ), light blue (NZO/HlLtJ), green (CAST/EiJ), red 689 (PWK/PhJ), and purple (WSB/EiJ) The horizontal dashed line on each plot corresponds to the 690 expected CN=2 state. Dashed black boxes highlight a small deletion and duplication in the Sox9 691 distal upstream regulatory region that are specific to the NOD/ShiLtJ haplotype. 692 693 694 Genetic Conflict mediated by Sex-linked Ampliconic Genes May Drive Sex Ratio 695 Distortion in a Subset of CC Strains 696 The Y-linked ampliconic gene Sly and its X-linked counterparts, Slx and Slxl1, are embroiled in 697 a genetic conflict over sex chromosome transmission during post-meiotic spermatogenesis 698 (Cocquet et al. 2012; Good 2012). Given that the CC founder strains include representatives 699 from three cardinal house mouse subspecies differing in their absolute Slx/Slxl1 and Sly copy 700 numbers (Morgan and Pardo-Manuel de Villena 2017), we hypothesized that Slx/Slxl1 - Sly 701 mediated conflict may underlie the pervasive pattern of SRD in this mapping population. 702 703 To address this possibility, we used publicly available whole genome sequences to estimate the 704 relative genomic copy number of these ampliconic genes in each realized CC strain (Table S15; 705 (Srivastava et al. 2017; Shorter et al. 2019b)). Slx/Slxl1 copy number varies approximately 2.5- 706 fold across strains (range: 24-64 copies), with only 5 strains exhibiting estimated haploid copy 707 number states that fall outside the range delimited by the parental genomes (parental range: 28- 708 60 haploid copies; Figure S10A). Across the CC population, Sly copy number ranges from 54- 709 168. CC founder whole genome sequences were generated from female samples, barring 710 comparisons of Sly copy number status in the parental inbred and realized CC strains. 711

712 713 Figure S10. (A) Slx/Slxl1 and Sly copy number estimates for the CC strains. Vertical dashed 714 lines denote estimated Slx/Slxl1 copy numbers for each of the 8 CC founder strains. The solid 715 black line is the least squares trend line fit to the data. (B) Sstx and Ssty1/2 copy number 716 estimates for the sequenced CC strains. As in (A), vertical dashed lines indicate the estimated

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717 Sstx copy number state of the inbred CC founder strains and the solid black line is an overall 718 trend line fit to the data using the method of least squares. Strains with significantly male- and 719 female-biased sex ratios are color-coded blue and red, respectively. 720 721 Overall, we find no correlation between the fraction of females at wean and Slx/Slxl1:Sly copy 722 number ratio (Spearman’s Rho = 0.0968, P = 0.490; Figure 5A). We validated the genomic 723 read-depth Slx/Slxl1:Sly estimated copy number (CN) ratios using ddPCR assays in a 724 representative subset of CC strains (Table S16). There is excellent qualitative alignment 725 between these two orthogonal methods for copy number estimation (Figure 5B; Spearman’s 726 Rho = 0.943, P = 0.0167), suggesting that the absence of a relationship between the 727 Slx/Slxl1:Sly ratio and SRD is unlikely due to technical errors. 728 729 Slx and Slxl1 are both neo-functionalized copies of SYCP3, but have rapidly diverged from each 730 other, exhibiting just 61% protein identity (Kruger et al. 2019). Recent experimental work 731 suggests that drive potential may be restricted to Slxl1: knockout of Slxl1, but not Slx, is 732 associated with a shift toward male-biased litters (Kruger et al. 2019). We find that the estimated 733 copy number of Slxl1, but not Slx, is positively correlated with SRD in the CC (Slxl1: 734 Spearman’s Rho: 0.334, P = 0.0146; Slx: Spearman’s Rho = 0.227, P = 0.102). However, the 735 ratio of Slxl1:Sly copy number carries no predictive association with SRD in this mapping 736 population (Spearman’s Rho = 0.105, P = 0.455), in contrast to expectations. 737 738 Despite the lack of an overall association between the Slx/Slxl1:Sly copy number ratio and sex 739 ratio distortion across CC strains, many sex biased strains do exhibit extreme amplicon copy 740 number ratios. In particular, CC006/TauUncJ has a relative excess of Sly copies relative to 741 Slx/Slxl1, consistent with the male bias in this strain. CC065/UncJ and CC058/UncJ, two 742 female-biased strains, exhibit a relative excess of Slx/Slxl1 compared to Sly, consistent with the 743 over-transmission of the X chromosome. However, there are clear exceptions to expected 744 trends. CC013/GeniUncJ has a moderately low Slx/Slxl1:Sly ratio, yet this strain is female- 745 biased. CC002/UncJ and CC028/GeniUncJ have high Slx/Slxl1:Sly ratios, in contrast to 746 predictions given the male sex bias observed in these strains (Figure 5A). 747 748 Recent molecular evidence suggests that SLX/SLXL1 and SLY compete for binding to SSTY1/2 749 and SPIN1, members of the spindlin gene family, to regulate expression at a large number of 750 genes expressed during spermatogenesis (Comptour et al. 2014; Kruger et al. 2019; Moretti et 751 al. 2020). SLY binding to SSTY1/2 or SPIN1 at gene promotors triggers the recruitment of the 752 SMRT/Ncor complex, repressing gene expression. In contrast, SLX and SLXL1 do not interact 753 with the SMRT/Ncor complex, and their association with SSTY1/2 at gene promoters leads to 754 the upregulation of target genes (Moretti et al. 2020). Ssty1/2 are Y-linked ampliconic genes, 755 whereas the mouse X-chromosome harbors several Spin1 gene clusters. The antagonistic 756 interactions of Ssty/Spin1 with Slx/Slxl1 and Sly prompted us to explore whether copy number 757 status at spindlin genes may factor into the complexity of SRD in the CC. 758 759 Spin1 and Ssty1/2 copy numbers span a 4.6- and 3.14-fold range in the CC population, 760 respectively (Spin1 range: 50-232 copies; Ssty1/2 range: 223-698 copies; Figure S10B). We 761 observe no significant relationship between the combined Spin1 and Ssty1/2 copy number and 762 sex ratio (Figure 5C; Spearman’s Rho = -0.178, P = 0.203). Based on the known interactions 763 between spindlins and members of the Sycp3-like family, we reasoned that the ratios of 764 Slx/Slxl1 to spindlin CN and Sly to spindlin CN might be correlated with the degree of SRD. 765 These predictions are not upheld (Spearman rank correlation P > 0.05; Figure S11). 766

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767 Overall, our findings uncover no global relationship between the copy number state of genes in 768 the Sycp3-like and spindlin gene families with SRD in the CC. However, the copy number state 769 of several CC lines accords with expectations under current models of SLX/SLXL1-SLY genetic 770 conflict, and we speculate that this established drive system may contribute to SRD in an 771 appreciable number of CC strains. Further, our genomic estimates of copy number for these 772 ampliconic genes may not accurately estimate the number of transcriptionally active genes in 773 each family. It is possible that a more widespread relationship between the copy number state 774 of these ampliconic genes and SRD is concealed by the inclusion of large numbers of non- 775 expressed pseudogenes in our copy number tallies. Lastly, we cannot rule out the likely 776 possibility that complex protein interactions between SLX/SLX1, SLY, spindlins and potentially 777 other ampliconic spermatid-expressed gene families contribute to the SRD documented in the 778 CC (Kruger et al. 2019; Moretti et al. 2020). 779

780 781 782 Figure 5. Relationship between copy number at ampliconic sex-linked genes and SRD. (A) 783 Correlation between the fraction of females at wean and the ratio of Slx/Slxl1:Sly gene copy 784 number. (B) Estimated copy numbers from genomic read depth and ddPCR are positively 785 correlated for members of the SYCP3-like gene family. (C) Correlation between the fraction of 786 females at wean and the copy number ratio of X:Y-linked genes in the spindlin gene family 787 (Sstx, Ssty1/2). Points corresponding to strains with significantly male- and female-biased sex 788 ratios are color-coded blue and red, respectively. 789

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790 791 792 Figure S11. (A) Relationship between the ratio of Slx/Slxl1 and spindlin copy numbers and SRD 793 and (B) the relationship between Sly and spindlin copy number and the fraction of females at 794 wean in the CC panel. Points corresponding to significantly sex-biased strains are color-coded 795 (blue = male-biased; red = female-biased). Horizontal dashed black line corresponds to a 796 balanced sex ratio of 0.5. 797 798 799 Many sex-biased CC strains exhibit reduced male fertility 800 SRD is frequently associated with reduced fertility in experimental crosses (Phadnis and Orr 801 2009; Cocquet et al. 2010; Meiklejohn et al. 2018; Zanders and Unckless 2019; Kruger et al. 802 2019), a trend that may emerge from the differential death, motility, or fertilization capacity of 803 sperm bearing one sex chromosome relative to the other. It is widely acknowledged that many 804 CC lines are poor breeders, and it appears that in most cases, reproductive output is 805 constrained by male fertility (Shorter et al. 2017). While there is no significant relationship 806 between average litter size and SRD among CC strains (Figure 3A), we sought to assess the 807 relationship between SRD and more precise measures of male fertility. 808 809 We accessed publicly available reproductive phenotype datasets for several CC strains from the 810 Mouse Phenome Database (Bogue et al. 2018; Table S6). Although the limited number of 811 phenotyped CC strains effectively bars a rigorous statistical analysis, many sex-biased strains 812 do appear to have reduced fertility relative to sex-balanced strains. Sex-biased CC strains tend 813 to have lower testis weights than non-sex-biased CC strains, although this association is not 814 statistically significant (Figure 6A; Spearman’s Rho = -0.382, P = 0.248). Similarly, despite the 815 lack of significant population-wide statistical correlations, several sex-biased strains - including 816 CC028/GeniUncJ, CC032/GeniUncJ, and CC040/TauUncJ – exhibit low fractions of motile 817 sperm and low sperm density compared to strains that yield sex-balanced litters (Figure 6B,C). 818 We conclude that many sex-biased strains exhibit phenotypic signatures of reduced male 819 fertility. 820

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821 822 Figure 6. Correlations between the magnitude of sex ratio distortion and (A) testis weight 823 standardized by body weight, (B) sperm density, (C) percentage of motile sperm, and (D) the 824 percentage of morphologically normal sperm. Dashed black lines trend lines were derived from 825 simple linear regression (y~x). 826 827 Mechanisms of SRD in CC032/GeniUncJ 828 CC032/GeniUncJ (hereafter, CC032) is the most extremely sex-biased CC strain, with only one 829 female out of every ~3 weaned pups. To our knowledge, SRD in this strain represents the 830 strongest reported departure from Mendelian expectations in a mammal. Our analyses of strain 831 breeding records indicate that SRD in CC032 cannot be entirely mediated by sex differences in 832 survival. CC032 has moderately-sized litters (4.2 pups/litter; 73rd percentile among CC strains) 833 and intermediate rates of neonatal mortality (20.6% mortality from birth to wean; 42nd percentile 834 among CC strains). Remarkably, even if all live-born CC032 pups that did not survive to wean 835 were female, this strain would still be significantly male-biased (Table S2; Binomial test P = 836 0.002565). 837 838 Relative to other CC lines, CC032 males have low average testis weights (Figure 6A), low 839 sperm density (Figure 6B), and reduced motility (Figure 6C). Histological analysis of testis 840 cross-sections reveal that CC032 males also have smaller seminiferous tubules than the

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841 majority of the CC founder strains (Figure 7A) and a higher fraction of tubules with vacuoles, 842 (Figures 7B-D). 843 844 Based on these phenotypic findings, we reasoned that targeted killing of X-bearing germ cells 845 could be a plausible explanation for the observed male bias in CC032. We used whole 846 chromosome painting to assess sex chromosome representation in mature sperm from this 847 strain. We observe equal numbers of X- and Y-bearing sperm (49.7% chrX-bearing sperm; 848 Binomial P = 0.854; Figures 7E and 7F), dismissing this explanation for SRD. 849

850 851 Figure 7. Reproductive phenotypes in CC032/GeniUncJ and the 8 CC founder strains. (A) 852 Mean tubule area in mm2 (+/- 1 standard deviation). (B) Representative stage VII/VIII tubule 853 cross section from CC032. (C) CC032 harbors a high fraction of tubules with large vacuoles. (D) 854 Percentage of tubules with vacuoles (+/- 1 standard deviation) for each of the 8 CC founders 855 and CC032. (E) Representative image of CC032 sperm hybridized with fluorescent paint probes 856 against chrY (green) and chrX (red). Y-bearing sperm carry a slight signal from chrX due to 857 shared X/Y homology across the pseudoautosomal region. (F) Fraction of X- and Y-bearing 858 sperm in CC032 from X and Y chromosome painting. 859 860 861 CC032 harbors a PWK Y-chromosome with a high Sly copy number and an X chromosome 862 bearing contributions from laboratory strains with M. m. domesticus ancestry (e.g., moderate 863 Slx/Slxl1 copy number). As a consequence of this sex chromosome haplotype structure, the

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864 ratio of Slx/Slxl1 to Sly in CC032 is lower than the CC-wide average (Figure 5A). The observed 865 Slx/Slxl1:Sly ratio in this strain is consistent with the observed male-bias. However, the strength 866 of SRD in CC032 exceeds that observed in both Slx/Slxl1 knockdown and knockout mice 867 (Cocquet et al. 2010; Kruger et al. 2019), suggesting the complementary action of other 868 mechanisms that exacerbate SRD on this genetic background. 869 870 We scanned the genome of CC032 for potential structural mutations in other sex-linked 871 ampliconic genes that could, conceivably, amplify the strength of Slx/Slxl1:Sly-mediated SRD. 872 Strikingly, the CC032 genome shows a pronounced enrichment of reads mapping to chrXA.1 873 (chrX:3-6Mb; Figure S12) and chrXqA3 (chrX:30.5-35.5 Mb; Figure 8). These loci harbor 874 clusters of Spin1 and Spin2, both members of the spindlin gene family, as well as a large 875 number of genes in the Btbd35f family. These regions encompass several gaps on the mm10 876 mouse reference assembly and are present at variable copy number across the 8 CC founder 877 strains. However, the read depth profile of CC032 at these loci exceeds what is observed in any 878 of the 8 founder strains (Figure 8; Figure S12). Several other CC lines exhibit similar read 879 depth patterns across these two X-linked loci, but intriguingly, the strain haplotype origin of 880 these amplified regions is variable (Figure 8). CC032 harbors C57BL/6J ancestry across both 881 regions, but strains with 129S1/SvImJ, NOD/ShiLtJ, and PWK/PhJ-derived haplotypes exhibit 882 near identical read-depth signatures (Figure 8; Figure S12). This observation would seem to 883 rule out a single common founder effect and imply an incredible rate of structural instability at 884 these loci. However, due to the complex, ampliconic architecture of these loci, we cannot 885 definitively rule out the possibility that assembly, mapping, or genotyping errors have led to mis- 886 assignment of strain haplotypes in these regions. Long-read sequence data for CC strains may 887 help resolve the architectural complexity of this locus and close standing assembly gaps. 888

889

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890 Figure 8. Estimated copy number state in the CC founder strains and 7 CC strains at chrX:30.5- 891 35.5 Mb. Dotted black lines correspond to CN=1. Tracks are color-coded by strain ancestry 892 (A/J: yellow, C57BL/6J: gray, 129S1/SvImJ: pink, NOD/ShiLtJ: dark blue, NZO/HlLtJ: light blue, 893 CAST/EiJ: green, PWK/PhJ: red, and WSB/EiJ: purple). Gaps in the read depth tracks 894 correspond to gaps in the mm10 reference genome assembly. CN states exceeding 10 are 895 clipped for visualization purposes. 896 897

898 899 900 Figure S12. Estimated copy number state in the CC founder strains and 7 CC strains at chrX:3- 901 6Mb. Dotted black lines correspond to CN=1. Tracks are color-coded by strain ancestry (A/J: 902 yellow, C57BL/6J: gray, 129S1/SvImJ: pink, NOD/ShiLtJ: dark blue, NZO/HlLtJ: light blue, 903 CAST/EiJ: green, PWK/PhJ: red, and WSB/EiJ: purple). Gaps in the read depth tracks 904 correspond to gaps in the mm10 reference genome assembly. CN states exceeding 10 are 905 clipped for visualization purposes. 906 907 Spindlins are chromatin readers that have been shown to directly bind to SLX and SLY 908 (Comptour et al. 2014; Kruger et al. 2019), although the consequences of this molecular 909 association are poorly understood. Very little is known about Btbd35f genes, but they are 910 regulated by SLX/SLXL1- and SLY in spermatids (Moretti et al. 2020). Our findings raise the 911 intriguing possibility that the extreme SRD in CC032 is mediated by a complex interplay 912 involving multiple sex-linked ampliconic gene families. However, further work is needed to

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449644; this version posted June 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

913 unravel the potential contributions of spindlins and Btbd35f to the dynamic chromatin 914 remodeling during post-meiotic spermatogenesis, and uncover how relative copy number 915 changes at these sex-linked ampliconic genes interface with mechanisms of Slx/Sly-mediated 916 SRD. 917 918 Sex Ratio Distortion in the Diversity Outbred Mouse Population 919 The Diversity Outbred (DO) population is a heterogeneous stock developed by outbreeding 920 early generation CC mice from distinct inbreeding funnels (Svenson et al. 2012). The population 921 is maintained as 175 outbred families defined by matrilineal inheritance. At every generation, a 922 female from family A is mated to a male from a randomly selected family B. Their progeny 923 comprise the next generation of DO lineage A. As a consequence of this breeding structure, DO 924 mice from a given family are more closely related than DO animals from different families. Thus, 925 if SRD has a genetic basis, males and females from individual DO lineages may sire an excess 926 of males or females relative to Mendelian expectations. 927 928 We used published DO breeding records to test for significant SRD in males and females from 929 each of the 175 DO families (Table S10; Chesler et al. 2016). Females from 15 DO families 930 have sex-biased litters (Binomial Test, uncorrected P < 0.05), with 10 families trending toward 931 an excess of males at wean (Figure 9a). Similarly, males from 12 DO lineages sire sex-biased 932 litters, with all but three producing an excess of males (Figure 9b). For both DO males and 933 females, the observed number of sex-biased families exceeds the ~9 families expected by 934 chance. Sample sizes within each DO lineage are modest (n=67-286; mean = 193), and we are 935 underpowered to detect slight departures from the expected sex ratio. Nonetheless, our findings 936 suggest that the 8-way genotypes associated with DO and CC mice are frequently associated 937 with SRD in both heterozygous and inbred states. 938 939 Despite an excess of both female and male DO lineages with significant SRD, there is no 940 correlation between the sex ratios of progeny sired by dams and sires from a given lineage 941 (Spearman’s Rho = -0.12; P = 0.1094; Figure S13). Indeed, there are no cases where DO 942 males and DO females from the same lineage both have sex-biased litters (Figure S13). 943 Evidently, the biological mechanisms of SRD in the DO (and, potentially, the CC) are largely 944 sex-limited in their manifestation. For instance, germline genetic conflict mediated by sex-linked 945 selfish elements only manifest effects on sex ratios via males whereas maternal effects are only 946 rendered through females. 947

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948 949 Figure 9. Sex ratios of weaned litters sired by (A) females and (B) males from each DO 950 breeding lineage. Lineages producing significantly male- and female-biased litters are color- 951 coded blue and red, respectively. Error bars correspond to 95% confidence intervals calculated 952 from the binomial distribution. 953

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Male Sex Ratio 954 955 Figure S13. Correlation between the sex ratio of litters sired by males and females from the 956 same DO breeding lineage. The outline color of each point denotes the direction of any 957 significant sex bias associated with male DO lineages. Conversely, the solid color of each point 958 indicates the direction of significant sex bias associated with female lineages. No lineages

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959 exhibit both male and female-associated sex bias. Blue: male bias; Red: female bias. Gray: no 960 significant sex bias.

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961 DISCUSSION 962 963 We have performed the first in-depth analysis of sex ratio distortion in the Collaborative Cross 964 multiparent mouse mapping population. Our integrated analyses of colony breeding records, 965 genome sequences, and phenotypic data uncover widespread SRD in the CC, with several 966 strains exhibiting extreme departures from sex ratio parity. These findings expose the complex 967 basis of sex ratio control, carry important implications for CC husbandry practices, and nominate 968 the CC as powerful resource for studying genetic conflict in action. 969 970 We show that CC strain sex ratios are stable over time, consistent across different breeding 971 facilities, and are not broadly dependent on maternal genotype or condition. However, we 972 acknowledge that our analyses are underpowered to detect weak temporal or maternal effects 973 on SRD. We are continuing to compile and analyze breeding records from the CC colony 974 maintained at The Jackson Laboratory, and the addition of new data over time will increase 975 statistical power and may allow detection of smaller effects. It is also critical to emphasize that 976 our investigations are limited to mice reared under a common set of standard laboratory 977 conditions. It remains possible that environmental perturbations or external stressors could 978 induce CC population-wide or strain-specific skews from the sex ratios reported here. Future 979 work is needed to explore the interaction of environmental variables with SRD in the CC. 980 Nonetheless, under the common housing conditions analyzed here, it appears that genetic 981 effects dominant other potential contributors to SRD in the CC population. 982 983 Multiple genetic mechanisms can give rise to SRD, including alleles that confer sex differences 984 in embryonic or neonatal survival or genetic mutations that induce sex reversal. We find no 985 consistent relationship between survival in early development and SRD in the CC. Similarly, our 986 analyses of structural variation at key genes in the sex determination pathway rule out sex 987 reversal as a likely explanation for the pervasive SRD in this mapping population. We also show 988 via QTL mapping that there are no large effect single-locus modifiers of SRD segregating in the 989 CC. These results, combined with the general absence of SRD in the inbred parental CC lines 990 and their F1 hybrids (Shorter et al. 2019a), imply that the underlying genetic mechanisms of 991 SRD are most likely attributable to multilocus combinations of alleles that are only revealed in 992 the 8-way CC population. 993 994 These considerations lead us to speculate that the SRD in this population arises, at least in part, 995 from the de-coupling of cryptic selfish sex-linked drive elements and their co-evolved 996 suppressors. Several lines of evidence lend support to this hypothesis. First, such genetic 997 conflicts necessary involve the interaction of multiple loci, aligning with the absence of SRD in 998 the CC founder strains and the lack of any large, single-locus effects in our QTL mapping. 999 Second, we observe minimal evidence for maternal effects and maternal genotype-dependence 1000 on SRD, implying that SRD in the CC is most frequently determined by mechanisms rendered 1001 through the male germline. Conflict between feuding drive elements on the X and Y 1002 chromosomes is, by genetic necessity, limited in expression to males. Third, transmission 1003 distorters typically act by disabling or killing non-carrier gametes, imposing a fitness cost to 1004 carriers (Zanders and Unckless 2019). Many sex-biased CC strains are characterized by small 1005 litter sizes (Figure 3a) and markers of reduced male fertility, including low testis weights, 1006 reduced sperm density, and impaired sperm motility (Figure 7). 1007 1008 One compelling candidate system is the SYCP3-like family of ampliconic sex-linked 1009 transmission distorters: Slx and Slxl1 on the X chromosome and their Y-linked paralog, Sly. 1010 Prior work has demonstrated that genetic imbalance of SLX/SLXL1 and SLY leads to disruption 1011 of the gene regulatory program in spermatids, sex ratio distortion, and infertility in house mice

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1012 (Cocquet et al. 2009, 2012; Kruger et al. 2019). The 8 CC founder strains differ in their native 1013 copy number status at these genes (Morgan and Pardo-Manuel de Villena 2017), and many 1014 sex-biased CC lines have inherited a relative excess or deficit of Slx/Slxl1 gene copies relative 1015 to Sly, consistent with their observed SRD. 1016 1017 However, we do not find a simple, overall relationship between SRD and Slx/Slxl1:Sly CN ratio 1018 across this population and several strains with extreme Slx/Slxl1:Sly ratios do not exhibit 1019 expected patterns of SRD (Figure 5A). Some genomic copies of these genes may be non- 1020 functional, and future work to quantify their mRNA or protein abundance in CC strains may help 1021 clarify the presumed underlying relationship between the protein products of these ampliconic 1022 genes and SRD. Beyond this possibility, recent work has begun to uncover the complexity of 1023 Slx/Slxl1 and Sly mediated conflict, suggesting that a simple linear relationship between gene 1024 copy number ratio and the magnitude of SRD is potentially overly simplistic. In particular, 1025 SLX/SLXL1 and SLY compete for binding to members of a second sex-linked amplicon gene 1026 family – the spindlin proteins SSTY1/2 and SPIN1 (Kruger et al. 2019; Moretti et al. 2020) – to 1027 antagonistically regulate large numbers of sex-linked and autosomal genes in post-meiotic 1028 spermatids. It is noteworthy that many sex-biased CC strains also have extreme spindlin 1029 genomic copy numbers (Figure 5C). Additionally, our focused investigations in 1030 CC032/GeniUncJ reveal apparent de novo expansions at two spindlin gene clusters on chrX, 1031 seemingly implicating these genes in the extreme SRD that characterizes this strain. We 1032 speculate that spindlins, and potentially other sex-linked ampliconic genes, interact with 1033 Slx/Slxl1 and Sly to modulate the strength of SRD in different CC strains. Future work to probe 1034 patterns of differential gene regulation in round spermatids from strains with extreme SRD 1035 versus those siring sex-balanced litters may help unlock the complex molecular mechanisms of 1036 SRD in this population. 1037 1038 Overall, the widespread trend of SRD across the CC and DO mouse populations seems to 1039 suggest that the intersubspecific 8-way genotypes segregating in these populations unmask a 1040 complex network of segregation distorters that are silenced in the context of individual inbred 1041 strains. Here, we focused on sex ratio distortion, as phenotypic sex provides a faithful readout of 1042 chromosome transmission (assuming no sex reversal). However, the high frequency of SRD 1043 across these populations raises the parallel prospect that selfish elements on other 1044 chromosomes could bias transmission in these 8-way diverse mouse populations. Indeed, a 1045 segregation distorter on chr2qC3 was previously identified via routine genetic monitoring in the 1046 DO population (Didion et al. 2015; Chesler et al. 2016). Specifically, the WSB/EiJ allele at this 1047 locus exhibits preferential segregation to the maternal oocyte during asymmetric female meiosis 1048 and threatened to drive to fixation, purging segregating variation at this locus from the DO. 1049 Although the DO maintenance breeding program is designed to minimize the potential for drive 1050 (Chesler et al. 2016), it is expected that, overtime, de novo evolution or the recombination of 1051 existing drive elements onto permissible genetic backgrounds could allow transmission 1052 distorters to take root. 1053 1054 Our findings also carry practical implications for CC strain maintenance and experimental 1055 design. Many CC strains exhibit only slight or no departure from Mendelian sex ratio 1056 expectations, but several yield strongly sex-biased litters. CC065/UncJ and CC032/GeniUncJ 1057 are the most notable examples, with just one male and one female in every three live-weaned 1058 pups, respectively. Given the modest reproductive output of most CC lines, these aspects of 1059 strain reproductive performance highlight the need for implementing strain-specific breeding 1060 programs to maintain stable colonies. Such practices are already in place at The Jackson 1061 Laboratory to ensure the long-term shelf-stability of this important diverse mouse resource, but 1062 should also be embraced in the settings of individual laboratories. Importantly, these breeding

34 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449644; this version posted June 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1063 challenges are not necessarily eliminated by outcrossing, as we document several CC-RIX 1064 backgrounds with SRD (Figure S6) and observe an excess of sex-biased lineages in the 1065 Diversity Outbred mapping population (Figure 9). 1066 1067 The CC population is an established resource for complex trait mapping and systems genetics 1068 investigation, and has yielded powerful new mouse models of human disease (Churchill et al. 1069 2004; Aylor et al. 2011; Philip et al. 2011; Rogala et al. 2014; Srivastava et al. 2017; Green et 1070 al. 2017). At the same time, the CC panel represents a pedigreed, well-resourced population 1071 optimally suited for investigations into the fundamental properties of genetic inheritance, 1072 including chromosome transmission. Each realized CC line harbors a unique multilocus 1073 combination of haplotypes from three cardinal house mouse subspecies, providing a real-time 1074 window into how intersubspecific allele permutations shape genome function and evolution. Our 1075 work has spotlighted the CC as a uniquely powerful platform for studying intragenomic genetic 1076 conflict and SRD in house mice and lays the groundwork for future investigations into the 1077 molecular basis of this important biological phenomenon. 1078 1079 1080 ACKNOWLEDGEMENTS 1081 We gratefully acknowledge the contribution of the Histopathology and Microscopy Scientific 1082 Services at The Jackson Laboratory for expert assistance with the work described in this 1083 publication. We are also indebted to Racheal Wallace for conserving cage cards from all 1084 Collaborative Cross mating units, enabling us to compile comprehensive breeding records from 1085 this mouse population. We thank Candice Baker and Cat Lutz for sharing breeding data from 1086 CC-RIX crosses. This work was funded by a NIGMS MIRA (R35GM133415) awarded to BLD. 1087 FB was supported by a Research Experience for Undergraduate Site Award (DBI-1262049; PI: 1088 Bob Braun).

35 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449644; this version posted June 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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